Biochem. J. (1988) 255, 153-159 (Printed in Great Britain) 153 Carnitine metabolism in the vitamin B-12-deficient rat Eric P. BRASS* and Sally P. STABLER Departments of Medicine and Pharmacology, Divisions of Clinical Pharmacology and Hematology, University of Colorado School of Medicine, Denver, CO 80262, U.S.A. In vitamin B-12 (cobalamin) deficiency the metabolism of propionyl-CoA and methylmalonyl-CoA are inhibited secondarily to decreased L-methylmalonyl-CoA mutase activity. Production of acylcarnitines provides a mechanism for removing acyl groups and liberating CoA under conditions of impaired acyl-CoA utilization. Carnitine metabolism was studied in the vitamin B-12-deficient rat to define the relationship between alterations in acylcarnitine generation and the development of methylmalonic aciduria. Urinary excretion of methylmalonic acid was increased 200-fold in vitamin B-12-deficient rats as compared with controls. Urinary acylcarnitine excretion was increased in the vitamin B-12-deficient animals by 700. This increase in urinary acylcarnitine excretion correlated with the degree of metabolic impairment as measured by the urinary methylmalonic acid elimination. Urinary propionylcarnitine excretion averaged 11 nmol/day in control rats and 120 nmol/day in the vitamin B- 12-deficient group. The fraction of total carnitine present as short-chain acylcarnitines in the plasma and liver of vitamin B- 12-deficient rats was increased as compared with controls. When the rats were fasted for 48 h, relative or absolute increases were seen in the urine, plasma, liver and skeletal-muscle acylcarnitine content of the vitamin B- 12-deficient rats as compared with controls. Thus vitamin B- 12 deficiency was associated with a redistribution of carnitine towards acylcarnitines. Propionylcarnitine was a significant constituent of the acylcarnitine pool in the vitamin B- 12-deficient animals. The changes in carnitine metabolism were consistent with the changes in CoA metabolism known to occur with vitamin B-12 deficiency. The vitamin B-12-deficient rat provides a model system for studying carnitine metabolism in the methylmalonic acidurias. INTRODUCTION data. Evidence obtained in vitro demonstrates that high concentrations of unusual organic acids such as Carnitine, in addition to its well-established role as an propionate can disrupt a number of metabolic pathways, obligate for the mitochondrial oxidation of long-chain including gluconeogenesis [7,8], fatty acid oxidation [8,9] fatty acids, interacts with other metabolic pathways by and pyruvate oxidation [8-11]. For propionate, many of generating acylcarnitines from the corresponding acyl- the cellular effects can be related to the accumulation of CoAs [1]. Under normal physiological conditions, the propionyl-CoA and methylmalonyl-CoA [12-15]. Under generation of short-chain acylcarnitines (acyl-group conditions where propionate interferes with cellular chain length less than 10 carbon atoms) from short-chain metabolism, addition of carnitine results in a partial acyl-CoAs operates near steady state [2,3], with the normalization of metabolism associated with a large distribution of total carnitine between free (unesterified) increase in propionylcarnitine production [8,9]. The use carnitine and the short-chain acylcarnitines reflecting the of carnitine in the treatment of patients with organic metabolic state of the tissue [3]. Under pathophysio- acidurias has resulted in clinical improvement of the logical conditions of acyl-CoA accumulation secondary patients and increased urinary excretion of the specific to a metabolic defect, the generation of acylcarnitines acylcarnitine corresponding to the accumulating acyl- provides a mechanism to remove the acyl group and CoA [4,5,16]. liberate free CoA. This reaction may be critical for The current studies were initiated to establish an maintaining normal cellular function under conditions animal model in vivo of impaired acyl-CoA utilization to where organic acids (and the corresponding acyl-CoAs) permit controlled studies of the inter-relationships accumulate [4]. This mechanism is thought to be in part between carnitine metabolism, the underlying metabolic responsible for the efficacy of carnitine in the treatment defect and overall cellular function. In the vitamin B-12 of some hereditary organic acidurias characterized (cobalamin)-deficient rat, the activity of L-methyl- by acyl-CoA accumulation secondary to an enzyme malonyl-CoA mutase is decreased [17]. This results in deficiency, such as in propionic acidaemia and the the secondary accumulation of propionyl-CoA and methylmalonic acidurias [4,5]. The resulting increase in methylmalonyl-CoA generated during the normal urinary acylcarnitine excretion may lead to insufficient metabolism of several amino acids and odd-chain-length endogenous carnitine to meet metabolic requirements fatty acids [18]. This situation is directly analogous to the [5,6]. human methylmalonic acidurias [19]. The current studies Understanding of the inter-relationship between the demonstrate that in the vitamin B-12-deficient rat there changes in endogenous carnitine metabolism, the is a marked increase in tissue short-chain acylcarnitine consequences of the acyl-CoA buildup and overall contents and in the urinary excretion of acylcarnitines, cellular function is limited by a lack of experimental including significant amounts of propionylcarnitine, * To whom all correspondence should be addressed. Vol. 255 154 E. P. Brass and S. P. Stabler Table 1. Compositions of the experimental diets Animals assigned to the vitamin B-12-deficient group were fed on an amino acid-based vitamin B-12-deficient The control and vitamin B- 12-deficient diets contained the diet (Teklad Inc., Madison, WI, U.S.A.), which was constituents listed below as determined by Teklad (g/kg of nutritionally complete except for the absence of vitamin diet): * indicates constituent in control diet only. B- 12 (Table 1). Food consumption for each rat given the deficient diet was recorded 3 times per week. On the basis L-Alanine 3.5 of this food consumption, a control rat was pair-fed L-Arginine hydrochloride 12.1 along with each rat in the vitamin B-12-deficient group. L-Asparagine 6.0 The control diet was identical with the deficient diet, L-Aspartic acid 3.5 except that vitamin B-12 was included (Table 1). All rats L-Cystine 3.5 were allowed water ad libitum. The rats were weighed L-Glutamic acid 40.0 approximately biweekly. Glycine 23.3 Urine was collected into a 50 ml Erlenmeyer flask L-Histidine hydrochloride,H20 4.5 L-Isoleucine 8.2 containing 10 ,tl of 1.2 M-HCI. The flask was held in a L-Leucine 11.1 container holding solid CO2, permitting the urine to be L-Lysine hydrochloride 18.0 kept frozen during a 24 h collection period. Animals L-Methionine 8.2 were killed by rapid decapitation, and blood was collected L-Phenylalanine 7.5 into chilled heparinized beakers. Plasma was prepared L-Proline 3.5 from the blood by centrifugation immediately after L-Serine 3.5 collection. Samples of liver and skeletal muscle were L-Threonine 8.2 obtained by rapid freeze-clamping of the tissue in L-Tryptophan 1.8 aluminium blocks cooled in solid C02/acetone. L-Tyrosine 5.0 L-Valine 8.2 Assays Sucrose 485.0 Corn starch 150.0 Carnitine was assayed by a modification of the Corn oil 100.0 radioenzymic assay of Cederblad et al. [20] as previously Cellulose 30.0 described [21]. Plasma and tissue samples were Calcium phosphate, dibasic 22.0 fractionated into acid-soluble and acid-insoluble Succinylsulphathiazol 10.0 fractions by precipitation in 3 % (v/v) HC104. The acid- Ethoxyquin 0.02 soluble fraction contained carnitine and short-chain Sodium chloride 2.59 acylcarnitines, and the acid-insoluble fraction contained Potassium citrate, monohydrate 7.7 long-chain acylcarnitines. Carnitine was generated from Potassium sulphate 1.82 acylcarnitines by alkaline hydrolysis and then quantified Magnesium oxide 0.84 Manganous carbonate 0.123 as carnitine in the radioenzymic assay. Acid-soluble Ferric citrate 0.210 fractions were analysed twice, once without hydrolysis as Zinc carbonate 0.056 a measure of carnitine, and again after hydrolysis to Cupric carbonate 0.011 measure the sum of carnitine and short-chain acyl- Potassium iodate 0.00035 carnitines (this sum is termed total acid-soluble carnitine). Sodium selenite 0.00035 Total carnitine refers to the sum of the carnitine, short- Chromium potassium sulphate 0.19 chain acylcarnitine and long-chain acylcarnitine p-Aminobenzoic acid (97.5 %) 0.110132 concentrations. Urine samples were analysed for Ascorbic acid 1.0166 carnitine and short-chain acylcarnitines by the same Biotin 0.000441 procedure, except that the HC104 fractionation was Calcium pantothenate 0.0661 Choline dihydrogen citrate 3.497 omitted. The carnitine concentration in carnitine Folic acid 0.002 standards used was determined by the method ofMarquis Inositol 0.1101 & Fritz [22]. Menadione 0.0496 Two techniques were used to study specific acyl- Niacin 0.0991 carnitines present in samples obtained. Fast-atom- Pyridoxine hydrochloride 0.022 bombardment mass spectrometry permitted assessment Riboflavin 0.022 of the acylcarnitine pool in samples and provided Thiamin hydrochloride 0.022 semi-quantitative information [9,23]; this analysis was Vitamin E acetate 0.0396 performed as previously described [9], with octanoyl- Vitamin A palmitate 0.0396 carnitine as an external standard. Acetylcarnitine Vitamin D3 0.0044 and propionylcarnitine were quantified by a reverse-